For the more than 40% of the world's people living in areas endemic for mosquito-borne diseases-as well as for those in emerging new areas-infectious diseases vectored by female mosquitoes continue to pose threats to health and life and to challenge researchers to find novel solutions to vector control and pathogen transmission. To meet this challenge, we propose to target the obligatory disease vector, the female mosquito, and to do so in a novel approach that eliminates the female mosquito at an immature stage before it can become a pathogen vector. Recently, we isolated, from an Aedes atropalpus hexamerin gene (Hex-1.2), specific enhancer-promoter regulatory DNA sequences, which we have termed the hexamerin-enhancer (Hex-Enh). The Hex-Enh targets gene activity both uniquely in females and to a critical insect tissue, the fat body (or liver), and is active primarily in late larvae and early pupae, i.e., immature, female mosquitoes. Thus, we hypothesize that the Hex-Enh constitutes a transcriptional regulatory module that can drive gene expression to destroy the female fat body and cause immediate death of immature female mosquitoes. We will test this hypothesis in Anopheles gambiae and Aedes aegypti by the following Specific Aims. Aim 1: Block development of, and kill, the immature female Ae. aegypti mosquito by using the Hex-Enh to target expression of a cell death gene (Michelob_x) to the female larval and pupal fat body. We will generate transgenic Ae. aegypti carrying 1) the tetracycline-repressible transcriptional activator gene (tTAV) under control of the Hex-Enh and 2) the Michelob_x gene as an effector gene to be induced exclusively in female larvae/pupae by tTAV. Using this inducible "tet-off" system engineered for mosquitoes, we expect to selectively kill female mosquito larvae and pupae by ablation of the fat body. Aim 2: Block development of, and kill, the immature female An. gambiae mosquito by using the Hex-Enh to target the expression of the homing endonuclease I-PpoI to the female larval and pupal fat body. I-PpoI expression is predicted to "shred" the X chromosome, resulting in the death of fat body cells. We will generate transgenic An. gambiae in which I-PpoI activity, directed by the Hex-Enh, in female larvae and pupae should result in ablation of the fat body and female death before adult eclosion. To date, no transgenic technology exists that can act as a female-specific mosquito larvicide. Our proposed killing of immature female mosquitoes can be applied to improve two important mosquito control strategies: Sterile Insect Technique (SIT), by developing novel genetic sexing strains and the release of only males;and "release of insects carrying a dominant lethal" (RIDL), by releasing males carrying a female-specific dominant lethal acting at the most effective preadult stage. If successful, our proposed development of novel technology for control of both major types of mosquitoes should dramatically reduce transmission of multiple infectious diseases vectored by either anopheline or culicine mosquitoes worldwide. PUBLIC HEALTH RELEVANCE: The worldwide resurgence of virulent and resistant forms of mosquito-borne diseases that kill millions of people each year has increased the urgency for novel strategies for mosquito control. This proposal focuses on improving and applying a molecular tool that will allow the development of a novel mosquito control strategy based on targeting the immature female mosquito, the actual disease vector. We will use the expression of transgenes specific for female mosquitoes at the late larval stage to improve the sterile insect technique (SIT) for insect control and to develop the means for driving transgenes into natural populations of insects.